Regulatory T cells (Treg) have emerged as a major mechanism in the maintenance of immunologic self-tolerance. Treg cells, which can emerge directly from the thymus (so called natural Treg cells) or be induced in the periphery (induced Treg cells) are usually CD4+CD25+ and function by inhibiting effector T cells. Elucidation of the mechanisms of Treg generation would be greatly facilitated by the identification of specific markers for Treg cells and molecules that mediate their function. We have been studying a self-tolerance system in which TCR transgenic CD4 T cells adoptively transferred into transgenic animals expressing the target antigen in multiple epithelial tissues become anergic and simultaneously develop into Treg cells capable of suppressing autoimmunity. Gene expression profiling of in vivo anergic/Treg cells compared with effector/memory T cells identified the LAG-3 gene as highly upregulated on anergic/Treg cells relative to effector/memory T cells. LAG-3hi Treg cells mediated suppression of T cell proliferative responses in an in vitro suppression system and anti-LAG-3 antibodies inhibited this suppression. These results identify LAG-3 as the first known specific cell surface marker for Treg cells that is potentially directly involved in mediating Treg function. This proposal seeks to further elucidate the mechanisms of suppression by LAG-3hi Treg cells and the specific role of the LAG-3 molecule in their activity. Specifically, we propose to: 1) Study the capacity and mechanisms of suppression mediated by Treg cells defined by expression of LAG-3 and CD25. 2) Elucidate the direct role of LAG-3 in modulating the activity of Treg cells and 3) Analyze the effect of blockade or elimination of LAG-3+ Treg cells in enhancing the activity of immunotherapies for established cancer.